Transgenic plant product development by conventional transformation and breeding efforts is a slow and unpredictable process. Gene targeting systems can overcome such problems as expression variability, unpredictable impacts of random gene insertion on agronomic performance, and the large number of experiments that need to be conducted to obtain ideal transgenic plants. Such systems can also provide approaches to manipulating endogenous genes.
Gene targeting systems require the ability to focus the recombination process to favor the recovery of desired targeting events. The natural cellular DNA repair and recombination machinery consists of a complex array of protein components interacting in a highly controlled manner to ensure that the fidelity of the genome is conserved throughout the many internal events or external stimuli experienced during each cell cycle. The ability to manipulate this machinery requires an understanding of how specific proteins are involved in the process, and how the genes that encode those proteins are regulated. Because many different protein components may be involved in gene targeting, the availability of host-specific genes and proteins could avoid possible problems of incompatibility associated with molecular interactions due to heterologous components.
The RAD23 gene of the budding yeast Saccharomyces cerevisiae is one of the 11 genes known to be involved in nucleotide excision repair (1, 2). Recent studies from several laboratories have also shown the requirement of RAD23 for the transcription-coupled repair as well as overall repair of DNA (3, 4, 5). Furthermore, the RAD23 gene product (denoted hereafter as Rad23) has also been implicated in ubiquitin mediated proteolysis (6) as well as cell cycle regulation (7). Rad23 is known to interact with a number of proteins involved in DNA repair, transcription, proteolysis and cell cycle to form separate, well defined higher order protein complexes, which in turn take part in the respective cellular events.
The regulation of the cell cycle and DNA/repair and recombination in plant systems by the modulation of maize Rad23 will provide improved and expanded methods of gene targeting and transformation. The need in the art for methods to regulate gene targeting and to increase transformation efficiency is clear. The present invention provides these and other advantages.